Neurogenetics of Stress Response and Reward Sensitivity For a number of years, we have been studying how spontaneous genetic variation that influences stress reactivity and/or reward sensitivity, which could be adaptive in certain contexts but may also increase risk for alcohol related problems. One of the first variants to be studied in our lab was the serotonin transporter linked polymorphism (HTTLPR), in which functional equivalents exist in humans and macaques. This variant is one that has been shown to interact with repeated, severe life stressors to predict depression, psychopathology and alcohol intake. We recently have shown that there are effects of genotype on responses to short-term maternal separation, with incremental increases in anxiety-like responses with repeated exposure. This suggests that HTTLPR genotype may have subtle effects on acute responses to stress (see Spinelli et al, 2013) that contribute to its cumulative effects on behavioral pathology, and that vulnerability to psychopathology can be moderated by genotype from a very early developmental stage. We have been studying how spontaneous genetic variation that influences reward sensitivity, and which could be adaptive in certain contexts, may also increase risk for alcohol related problems. In both humans and rhesus macaques, there are polymorphisms in the mu-opioid receptor gene (OPRM1) that influence affinity of the receptor for its endogenous ligand, -Endorphin. We and others have shown that these polymorphisms predict increased alcohol-induced euphoria and decreased stress response, suggesting them to have gain-of-function roles. We also have demonstrated it to relate to individual differences in response to natural rewards, as shown by measures of both reward bias in humans (Lee et al, 2012) and social attachment in both human and macaque (Barr et al, 2008; Higham et al, 2011; Copeland et al, 2011). Dopamine neurotransmission underlies many reward-dependent and reinforcing processes. Repeat polymorphisms in the DRD4 gene exist across a variety of animal species (humans, macaques, vervets, dogs, horses, and chimpanzees) and have in some instances been shown to predict behaviors related to altered sensitivity to reward. Macaque species also exhibit variation at this gene (pigtail macaques and Tonkean macaques), which differ in frequencies across species. One trait predicted by DRD4 variation in humans is that of novelty seeking. This has been replicated in vervet monkeys, in which a DRD4 length variant was also observed. Of interest, in the dog, which has been subject to intense artificial selection, a repeat polymorphism predicts social impulsivity and activity-impulsivity endophenotypes. We are currently sequencing across primate datasets to identify length variation at this locus. We have confirmed its existence in rhesus macaques and are screening other species as well. We are also genotyping human and rhesus datasets and will be performing genotype-phenotype correlations across species to see if DRD4 genotype predicts variation in the traits of novelty seeking, reward sensitivity and alcohol consumption. Identification of Genetic Variation in Rhesus Macaques using Massively Paralleled Sequencing In collaboration with the Section of Human Neurogenetics, we performed ChIP- and RNA-SEQ using archived brain from rhesus macaques reared under both normal and stressful conditions. While fewer than 1,000 rhesus SNPs had been known, we identified more than 500,000 SNPs using this gene-centric approach. Further, using the equivalent methodology in human, we were able to perform a comparative analysis. This revealed that the rhesus macaque, which is a widespread, highly adaptable species, is approximately three times as diverse as the human but more closely equivalent in damaging variation (Yuan et al, 2012). We recently sequenced the exomes of rhesus macaque subjects that were selected based on variation in temperament. The advantages exome sequencing this approach are that DNA can easily be obtained from peripheral tissues (such as blood, hair follicles or skin) and that there is higher potential for identifying functional variants, since only the protein encoding sequences are obtained. We identified non-synonymous SNPs (variants that predicted amino acid changes), some of which were predicted to be damaging by in silico analysis. Among these were SNPs in the GABARAA6 and CRHR1 genes, which encode the alpha-6 subunit of the GABA-A receptor and the corticotropin releasing hormone receptor. Variation at these genes would be predicted to relate to variation in anxious responding and alcohol response. The occurrence of these variants is presently being verified using traditional sequencing methods. Other primate samples have been obtained for performing similar studies across species.